Accessible Requires Authentication Published by De Gruyter August 8, 2005

Inhibition of aldosterone biosynthesis by staurosporine

Matthias Bureik, Alexander Mion, Christopher J. Kenyon and Rita Bernhardt
From the journal


Staurosporine (STS) is a very potent broad-range kinase inhibitor, and its antiproliferative properties made it a lead compound for protein kinase C (PKC) inhibitors with therapeutic potential. Because STS also causes hypotension, we investigated in this study whether it directly interferes with the terminal steps of aldosterone biosynthesis; these are catalysed by a mitochondrial steroid hydroxylase system consisting of adrenodoxin reductase, adrenodoxin, and the cytochrome P450 enzyme hCYP11B2 (aldosterone synthase). Here we demonstrate that nanomolar concentrations of STS significantly reduced aldosterone synthase activity in transiently transfected COS-1 cells and in stably transfected V79MZh11B2 cells (IC50=11 nM). However, STS did not inhibit bovine aldosterone synthase in a reconstituted steroid hydroxylation assay. In transiently transfected COS-1 cells, the protein level of adrenodoxin (but not that of adrenodoxin reductase or of hCYP11B2) was significantly reduced after treatment with 2 nM STS. Finally, we show that STS treatment (1 μg/day) of mice reduced their aldosterone/renin ratio by almost 50% (p=0.015). To the best of our knowledge, this is the first report of a direct in vivo effect of STS on the renin-angiotensin-aldosterone system. We conclude (i) that the hypotensive effect of staurosporine is at least partly due to inhibition of aldosterone biosynthesis via adrenodoxin depletion, and (ii) that aldosterone biosynthesis can be regulated in vivo at the level of adrenodoxin availability.


Corresponding author


Abe, K., Yoshida, M., Usui, T., Horinouchi, S., and Beppu, T. (1991). Highly synchronous culture of fibroblasts from G2 block caused by staurosporine, a potent inhibitor of protein kinases. Exp. Cell Res.192, 122–127. Search in Google Scholar

Bechtel, S., Belkina, N., and Bernhardt, R. (2002). The effect of amino-acid substitutions I112P, D147E and K152N in CYP11B2 on the catalytic activities of the enzyme. Eur. J. Biochem.269, 1118–1127. Search in Google Scholar

Bernhardt, R. (1996). Cytochrome P450: structure, function, and generation of reactive oxygen species. Rev. Physiol. Biochem. Pharmacol.127, 137–221. Search in Google Scholar

Bernhardt, R. (2000). The role of adrenodoxin in adrenal steroidogenesis. Curr. Opin. Endocrin. Diab.7, 109–115. Search in Google Scholar

Bilder, G.E., Kasiewski, C.J., and Perrone, M.H. (1990). Phorbol-12,13-dibutyrate-induced vasoconstriction in vivo: characterization of response in genetic hypertension. J. Pharmacol. Exp. Ther.252, 526–530. Search in Google Scholar

Bruno, S., Ardelt, B., Skierski, J.S., Traganos, F., and Darzynkiewicz, Z. (1992). Different effects of staurosporine, an inhibitor of protein kinases, on the cell cycle and chromatin structure of normal and leukemic lymphocytes. Cancer Res.52, 470–473. Search in Google Scholar

Bureik, M., Zeeh, A., and Bernhardt, R. (2002). Modulation of steroid hydroxylase activity in stably transfected V79MZh11B1 and V79MZh11B2 cells by PKC and PKD inhibitors. Endocr. Res.28, 351–355. Search in Google Scholar

Bureik, M., Zöllner, A., Schuster, N., Montenarh, M., and Bernhardt, T. (2005). Phosphorylation of bovine adrenodoxin by protein kinase CK2 affects the interaction with its redox partner cytochrome P450scc (CYP11A1). Biochemistry44, 3821–3830. Search in Google Scholar

Cao, P.R. and Bernhardt, R. (1999). Interaction of CYP11B1 (cytochrome P-45011 β) with CYP11A1 (cytochrome P-450scc) in COS-1 cells. Eur. J. Biochem.262, 720–726. Search in Google Scholar

Crissman, H.A., Gadbois, D.M., Tobey, R.A., and Bradbury, E.M. (1991). Transformed mammalian cells are deficient in kinase-mediated control of progression through the G1 phase of the cell cycle. Proc. Natl. Acad. Sci. USA88, 7580–7584. Search in Google Scholar

Curnow, K.M., Tusie-Luna, M.T., Pascoe, L., Natarajan, R., Gu, J.L., Nadler, J.L., and White, P.C. (1991). The product of the CYP11B2 gene is required for aldosterone biosynthesis in the human adrenal cortex. Mol. Endocrinol.5, 1513–1522. Search in Google Scholar

Denner, K. and Bernhardt, R. (1998). Inhibition studies of steroid conversions mediated by human CYP11B1 and CYP11B2 expressed in cell cultures. In: Oxygen Homeostasis and its Dynamics, K. Ishimura, H. Shimada and M. Suematsu, eds. (Tokyo, Japan: Springer Tokyo Inc.), pp. 231–236. Search in Google Scholar

Denner, K., Doehmer, J., and Bernhardt, R. (1995). Cloning of CYP11B1 and CYP11B2 from normal human adrenal and their functional expression in COS-7 and V79 Chinese hamster cells. Endocr. Res.21, 443–448. Search in Google Scholar

Ehmer, P.B., Bureik, M., Bernhardt, R., Muller, U., and Hartmann, R.W. (2002). Development of a test system for inhibitors of human aldosterone synthase (CYP11B2): screening in fission yeast and evaluation of selectivity in V79 cells. J. Steroid Biochem. Mol. Biol.81, 173–179. Search in Google Scholar

Gescher, A. (2000). Staurosporine analogues – pharmacological toys or useful antitumour agents? Crit. Rev. Oncol. Hematol.34, 127–135. Search in Google Scholar

Krohn, A.J., Preis, E., and Prehn, J.H. (1998). Staurosporine-induced apoptosis of cultured rat hippocampal neurons involves caspase-1-like proteases as upstream initiators and increased production of superoxide as a main downstream effector. J. Neurosci.18, 8186–8197. Search in Google Scholar

Lever, M.A., Th'ng, J.P., Sun, X., and Hendzel, M.J. (2000). Rapid exchange of histone H1.1 on chromatin in living human cells. Nature408, 873–876. Search in Google Scholar

Lifton, R.P., Dluhy, R.G., Powers, M., Rich, G.M., Cook, S., Ulick, S., and Lalouel, J.M. (1992). A chimeric 11-β-hydroxylase/aldosterone synthase gene causes glucocorticoid-remediable aldosteronism and human hypertension. Nature355, 262–265. Search in Google Scholar

MacPhee, I.A., Antoni, F.A., and Mason, D.W. (1989). Spontaneous recovery of rats from experimental allergic encephalomyelitis is dependent on regulation of the immune system by endogenous adrenal corticosteroids. J. Exp. Med.169, 431–445. Search in Google Scholar

Millar, J.A., Leckie, B.J., Morton, J.J., Jordan, J., and Tree, M. (1980). A microassay for active and total renin concentration in human plasma based on antibody trapping. Clin. Chim. Acta101, 5–15. Search in Google Scholar

Monnier, N., Defaye, G., and Chambaz, E.M. (1987). Phosphorylation of bovine adrenodoxin. Structural study and enzymatic activity. Eur. J. Biochem.169, 147–153. Search in Google Scholar

Mornet, E., Dupont, J., Vitek, A., and White, P.C. (1989). Characterization of two genes encoding human steroid 11-beta-hydroxylase (P-450(11) β). J. Biol. Chem.264, 20961–20967. Search in Google Scholar

Nemani, R., Ghazarian, J.G., Moorthy, B., Wongsurawat, N., Strong, R., and Armbrecht, H.J. (1989). Phosphorylation of ferredoxin and regulation of renal mitochondrial 25-hydroxyvitamin D-1 α-hydroxylase activity in vitro. J. Biol. Chem.264, 15361–15366. Search in Google Scholar

Okamura, T., John, M.E., Zuber, M.X., Simpson, E.R., and Waterman, M.R. (1985). Molecular cloning and amino acid sequence of the precursor form of bovine adrenodoxin: evidence for a previously unidentified COOH-terminal peptide. Proc. Natl. Acad. Sci. USA82, 5705–5709. Search in Google Scholar

Okazaki, T., Kato, Y., Mochizuki, T., Tashima, M., Sawada, H., and Uchino, H. (1988). Staurosporine, a novel protein kinase inhibitor, enhances HL-60-cell differentiation induced by various compounds. Exp. Hematol.16, 42–48. Search in Google Scholar

Okuda, T., Sawada, H., Kato, Y., Yumoto, Y., Ogawa, K., Tashima, M., and Okuma, M. (1991). Effects of protein kinase A and calcium/phospholipid-dependent kinase modulators in the process of HL-60 cell differentiation: their opposite effects between HL-60 cell and K-562 cell differentiation. Cell Growth Differ.2, 415–420. Search in Google Scholar

Omura, S., Iwai, Y., Hirano, A., Nakagawa, A., Awaya, J., Tsuchya, H., Takahashi, Y., and Masuma, R. (1977). A new alkaloid AM-2282 of Streptomyces origin. Taxonomy, fermentation, isolation and preliminary characterization. J. Antibiot. (Tokyo)30, 275–282. Search in Google Scholar

Omura, S., Sasaki, Y., Iwai, Y., and Takeshima, H. (1995). Staurosporine, a potentially important gift from a microorganism. J. Antibiot. (Tokyo)48, 535–548. Search in Google Scholar

Sausville, E.A., Arbuck, S.G., Messmann, R., Headlee, D., Bauer, K.S., Lush, R.M., Murgo, A., Figg, W.D., Lahusen, T., Jaken, S., Jing, X., Roberge, M., Fuse, E., Kuwabara, T., and Senderowicz, A.M. (2001). Phase I trial of 72-h continuous infusion UCN-01 in patients with refractory neoplasms. J. Clin. Oncol.19, 2319–2333. Search in Google Scholar

Schwartz, G.K., Redwood, S.M., Ohnuma, T., Holland, J.F., Droller, M.J., and Liu, B.C. (1990). Inhibition of invasion of invasive human bladder carcinoma cells by protein kinase C inhibitor staurosporine. J. Natl. Cancer Inst.82, 1753–1756. Search in Google Scholar

Shizuta, Y., Kawamoto, T., Mitsuuchi, Y., Miyahara, K., Rosler, A., Ulick, S., and Imura, H. (1995). Inborn errors of aldosterone biosynthesis in humans. Steroids60, 15–21. Search in Google Scholar

Siegel, N., Wongsurawat, N., and Armbrecht, H.J. (1986). Parathyroid hormone stimulates dephosphorylation of the renoredoxin component of the 25-hydroxyvitamin D3-1α-hydroxylase from rat renal cortex. J. Biol. Chem.261, 16998–17003. Search in Google Scholar

Solish, S.B., Picado-Leonard, J., Morel, Y., Kuhn, R.W., Mohandas, T.K., Hanukoglu, I., and Miller, W.L. (1988). Human adrenodoxin reductase: two mRNAs encoded by a single gene on chromosome 17cen→q25 are expressed in steroidogenic tissues. Proc. Natl. Acad. Sci. USA85, 7104–7108. Search in Google Scholar

Th'ng, J.P., Guo, X.W., Swank, R.A., Crissman, H.A., and Bradbury, E.M. (1994). Inhibition of histone phosphorylation by staurosporine leads to chromosome decondensation. J. Biol. Chem.269, 9568–9573. Search in Google Scholar

Toledo, L.M. and Lydon, N.B. (1997). Structures of staurosporine bound to CDK2 and cAPK – new tools for structure-based design of protein kinase inhibitors. Structure5, 1551–1556. Search in Google Scholar

Ulmschneider, S., Müller-Vieira, U., Mitrenga, M., Hartmann, R.W., Oberwinkler-Marchais, S., Klein, C.D., Bureik, M., Bernhardt, R., Antes, I., and Lengauer, T. (2005). Synthesis and evaluation of imidazolylmethylene-tetrahydronaphthalenes and -indanes: potent inhibitors of aldosterone synthase (CYP11B2). J. Med. Chem.48, 1796–1805. Search in Google Scholar

Utz, I., Hofer, S., Regenass, U., Hilbe, W., Thaler, J., Grunicke, H., and Hofmann, J. (1994). The protein kinase C inhibitor CGP 41251, a staurosporine derivative with antitumor activity, reverses multidrug resistance. Int. J. Cancer57, 104–110. Search in Google Scholar

Wolff, T., Distlerath, L.M., Worthington, M.T., Groopman, J.D., Hammons, G.J., Kadlubar, F.F., Prough, R.A., Martin, M.V., and Guengerich, F.P. (1985). Substrate specificity of human liver cytochrome P-450 debrisoquine 4-hydroxylase probed using immunochemical inhibition and chemical modeling. Cancer Res.45, 2116–2122. Search in Google Scholar

Yasoshima, K., Kuwabara, T., Fuse, E., Kuramitu, T., Kurata, N., Nishiie, H., Oishi, T., Kobayashi, H., and Kobayashi, S. (2001). Pharmacokinetics, distribution, metabolism and excretion of [3H]UCN-01 in rats and dogs after intravenous administration. Cancer Chemother. Pharmacol.47, 106–112. Search in Google Scholar

Ye, P., Kenyon, C.J., Mackenzie, S.M., Seckl, J.R., Fraser, R., Connell, J.M., and Davies, E. (2003). Regulation of aldosterone synthase gene expression in the rat adrenal gland and central nervous system by sodium and angiotensin II. Endocrinology144, 3321–3328. Search in Google Scholar

Yue, T.L., Wang, C., Romanic, A.M., Kikly, K., Keller, P., Dewolf, W.E. Jr., Hart, T.K., Thomas, H.C., Storer, B., Gu, J.L., Wang, X., and Feuerstein, G.Z. (1998). Staurosporine-induced apoptosis in cardiomyocytes: a potential role of caspase-3. J. Mol. Cell. Cardiol.30, 495–507. Search in Google Scholar

Published Online: 2005-08-08
Published in Print: 2005-07-01

©2005 by Walter de Gruyter Berlin New York